Aspiration-free well plate apparatus and methods

ABSTRACT

A well plate includes a including a top portion, a bottom portion and a membrane disposed between the top portion and the bottom portion. The top portion defines a sample well in fluid communication with an opening defined by the membrane and in fluid communication with a reservoir defined by the bottom portion. The well plate is configured to be used in a centrifugation process of a test sample including a sample material and a wash liquid. The test sample configured to be received within the sample well and the reservoir. The membrane configured to filter the wash liquid from the test sample during the centrifugation process such that the wash liquid can pass from the reservoir, through the membrane and can be captured within a collection chamber while the sample material remains within the reservoir.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/635,386, entitled “Aspiration-Free Well Plate Apparatus and Methods,”filed Mar. 2, 2015, which is a divisional of U.S. patent applicationSer. No. 14/215,645, entitled “Aspiration-Free Well Plate Apparatus andMethods,” filed Mar. 17, 2014 (now U.S. Pat. No. 8,968,682), whichclaims priority to and the benefit of U.S. Provisional PatentApplication No. 61/787,554, entitled “Aspiration-Free Well PlateApparatus and Methods,” filed Mar. 15, 2013, each of the disclosures ofwhich is incorporated herein by reference in its entirety.

BACKGROUND

Embodiments described herein relate generally to biological testingdevices and more particularly, to apparatus and methods of using testplates having multiple wells for holding multiple samples to be used inanalytical testing fields.

Cellular assays typically require exposing cells to a series ofdifferent liquids with different properties, usually referred to asculture media, buffers, stains, staining cocktails, fixatives,permeabilization agents, and similar liquids. The cells are exposed to aseries of different liquids by removing the cells from the majority ofthe current liquid and then adding the next successive liquid in theseries. This is typically done in the following manner: cells suspendedin their current liquid are transferred to a tube or to the wells of awell plate, and then spun in a centrifuge at a speed sufficient togenerate a centripetal acceleration that can pull the cells to thebottom of the well (e.g., between about 200 relative centrifugal force(rcf) and about 600 rcf) to form what is known as a “pellet”. The liquid(supernatant) can then be decanted or aspirated and discarded, leavingthe cell pellet at the bottom of the tube or well in a relatively smallresidual volume of the liquid. The next liquid in the series can then beadded into a volume that is much larger than the residual volume of thelast liquid, and as such, the cells can be exposed almost exclusively tothe new liquid.

A known problem with this approach, however, is that a fraction of thecells can be lost each time the sample is centrifuged and the liquidfraction removed by decanting or aspiration. This problem is compoundedby starting with a very small cell number (e.g., fewer than 200,000cells) because the stability of the pellet is proportional to the numberof cells in the pellet. For example, at low cell numbers the pelletresulting from centrifugation is unstable and an unacceptably largefraction of the sample is lost when the supernatant is discarded bydecanting or aspiration. As many experiments may require five or moreserially performed centrifugation steps, with some requiring more thantwenty centrifugation steps, it may be impractical to work with low cellnumbers if a high fraction of the starting cell number must be recoveredat the end of all of the centrifugation steps. This is problematic forthe growing number of applications involving limited sample material,including analysis of biological samples from pediatric patients orcells derived from certain biopsies or clinical aspirates. Inparticular, primary stem cells or derived stem cells are usuallyavailable in very small numbers and the quantity that can be used in anassay may be as low as 5,000 cells, or lower. Conventionalcentrifugation and aspiration or decanting approaches are poorly suitedfor cell numbers as low as 5,000 as, in some instances, effectively noneof the cells can be recovered. Many cell types, and stem cells inparticular, are sensitive to the high centripetal acceleration forcerequired by conventional methods.

An alternative approach has been developed for bead-based assays wherethe bottom of the wells of a well plate include a porous membrane. Suchwell plates can be centrifuged or placed on a manifold that uses avacuum to pull the liquid fraction through the bottom of the plate.Unfortunately, when cells are used in these membrane-bottomed cellplates a high fraction of the cells become irretrievably stuck in themembrane material, even when using state-of-the-art low-bindingmembranes making this unsuitable for many downstream cellular assays.

An additional problem of the conventional methods mentioned above isthat the supernatant that is separated from the cells may containbiohazardous elements, such as infectious viruses like Hepatitis, HIV,or other agents. Conventional methods decant the supernatant or aspiratethe supernatant to transfer it to a vessel that contains chemicals thatneutralize the biohazardous elements. The process of decanting oraspirating of the supernatant carries a risk of exposing the personperforming the assay to the biohazards in the supernatant. Eliminatingthe need to decant or aspirate the supernatant can reduce this risk.

Thus, there is a need for an effective way to repeatedly wash cellswithout substantially losing cells in each wash step. In addition, thereis a need for a way to effectively deal with a biohazardous supernatantby minimizing handling. Also, there is a need for an effective way toreduce centrifugation forces on the cells to increase cell viability,while still maintaining high sample separation and recovery.

SUMMARY

Apparatus and methods of using test tubes or plates having one or morewells (e.g., multi-well plates) for holding one or more samples to beused in analytical testing fields are described herein. In someembodiments, a well plate includes a top portion, a bottom portion and amembrane disposed between the top portion and the bottom portion. Thetop portion defines a sample well in fluid communication with an openingdefined by the membrane and in fluid communication with a reservoirdefined by the bottom portion. The well plate is configured to be usedin a centrifugation process of a test sample including a sample materialand a wash liquid. The test sample is configured to be received withinthe sample well and the reservoir. The membrane is configured to filterthe wash liquid from the test sample during the centrifugation processsuch that the wash liquid can pass from the reservoir, through themembrane and can be captured within a collection chamber, while thesample material remains within the reservoir.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a known well plate with a manifold fordrawing liquid through the bottom of the well plate.

FIG. 2 is a perspective view of a well plate assembly, according to anembodiment, with a portion of the well plate of the plate assemblyremoved to show inner details.

FIG. 3 is an enlarged perspective view of a portion of the well plateassembly of FIG. 2.

FIG. 4 is a detailed cut-away view of a well of the well plate assemblyof FIG. 2.

FIGS. 5-8 are a top view, a bottom view, a right side view, and a frontview, respectively, of the top plate of FIG. 2.

FIGS. 9 and 10 are a perspective view and a top view, respectively, ofthe top plate of FIG. 2.

FIG. 11 is a cross-sectional view of a portion of the top plate takenalong line B-B in FIG. 10.

FIG. 12 is a top view of a base plate of the well plate assembly of FIG.2; and

FIG. 13 is a cross-sectional view of the base plate taken along line-A-Ain FIG. 12.

FIG. 14 is a top view of a membrane of the well plate assembly of FIG.2.

FIG. 15 is an exploded view of a well plate assembly, according toanother embodiment.

FIG. 16 is a detailed cross-sectional view of a portion of the wellplate assembly of FIG. 15.

FIG. 17 is a perspective view of a well plate assembly, according to anembodiment and an effluent tray, according to an embodiment.

FIG. 18 is a top perspective view of the effluent tray of FIG. 17.

FIG. 19 is a perspective view of the well plate assembly of FIG. 17.

FIG. 20 is perspective view of a well plate assembly, according toanother embodiment.

FIG. 21 is an exploded view of the well plate assembly of FIG. 20 andthe effluent tray of FIG. 18.

FIG. 22 is a top view of a membrane of the well plate assembly of FIG.20.

FIG. 23 is a perspective view of a well plate assembly, according toanother embodiment.

FIG. 24 is an exploded view of the well plate assembly of FIG. 23.

FIG. 25 is an enlarged view of a portion of the well plate assembly ofFIG. 23 with a portion of the well plate assembly removed forillustration purposes.

FIG. 26 is a perspective view of a tube assembly, according to anembodiment, with a portion of the tube assembly removed for illustrationpurposes.

FIG. 27 is a perspective view of the tube assembly of FIG. 26, with aportion of the tub assembly removed for illustration purposes.

FIG. 28 is an exploded view of the tube assembly of FIG. 26.

FIG. 29 illustrates multiple flow cytometry dot plots of example datagenerated using, for example, a well plate assembly according to anembodiment, showing proof-of-principle and efficacy.

FIGS. 30 and 31 each illustrate different plots of flow cytometry datagenerated using, for example, a well plate assembly according to anembodiment, showing proof-of-principle and efficacy.

FIGS. 32 and 33 each show flow cytometry plots of example data generatedusing, for example, a well plate assembly according to an embodiment,showing proof-of-principle and efficacy.

FIG. 34 is a perspective view of a well plate assembly and a trayassembly according to another embodiment.

FIG. 35 is a partially exploded view of the tray assembly and well plateassembly of FIG. 34.

FIGS. 36 and 37 are exploded views of a first portion and a secondportion, respectively, of the tray assembly of FIG. 34.

FIG. 38 is a perspective view of a top plate included in the well plateassembly of FIG. 34.

FIG. 39 is a perspective view of a membrane included in the well plateassembly of FIG. 34.

FIG. 40 is a perspective view of a bottom plate included in the wellplate of FIG. 34.

FIG. 41 is a cross-sectional view of the well plate assembly of FIG. 34,taken along the line C-C in FIG. 34.

FIG. 42 is a perspective view of a well plate assembly and a trayassembly according to another embodiment.

FIG. 43 is a partially exploded view of the well plate assembly and thetray assembly of FIG. 42.

FIG. 44 is a cutaway view of a portion of the well plate assembly and aportion of the tray assembly of FIG. 42.

FIG. 45 is a perspective view of the well plate assembly and the trayassembly of FIG. 42 being coupled to a lid.

FIG. 46 is an exploded view of the well plate assembly of FIG. 42.

FIG. 47 is a cross-sectional view of the well plate assembly in FIG. 46in an exploded configuration.

FIGS. 48 and 49 are top views of a top plate and a bottom plate,respectively, included in the well plate assembly of FIG. 42.

FIG. 50 is a cross-sectional side view of the bottom plate included inthe well plate assembly of FIG. 42.

FIG. 51 is cross-sectional view of the well plate assembly in FIG. 46.

FIGS. 52 and 53 are top views of a top plate and a bottom plate,respectively, included in a well plate assembly, according to anembodiment.

FIG. 54 is a cross-sectional side view of the bottom plate included inthe well plate assembly of FIGS. 52 and 53.

FIGS. 55 and 56 are top views of a top plate and a bottom plate,respectively, included in a well plate assembly, according to anembodiment.

FIG. 57 is a cross-sectional side view of the bottom plate included inthe well plate assembly of FIGS. 55 and 56.

FIGS. 58 and 59 are a top view and a bottom view, respectively, of a topplate included in a well plate assembly, according to an embodiment.

FIG. 60 is a cross-sectional side view of a bottom plate included in thewell plate assembly of FIGS. 58 and 59.

FIGS. 61 and 62 are top views of top plates included in different wellplate assemblies, each according to different embodiments.

FIG. 63 is an exploded view of a well plate assembly according to anembodiment.

FIG. 64 is a top view of the well plate assembly of FIG. 63.

FIG. 65 is a cross-sectional view of the well plate assembly of FIG. 64taken along the line D-D.

DETAILED DESCRIPTION

Apparatus and methods of using test tubes or plates having one or morewells (e.g., multi-well plates) for holding one or more samples to beused in analytical testing fields are described herein. In someembodiments, a well plate includes a top portion, a bottom portion and amembrane disposed between the top portion and the bottom portion. Thetop portion defines a sample well in fluid communication with an openingdefined by the membrane and in fluid communication with a reservoirdefined by the bottom portion. The well plate is configured to be usedin a centrifugation process of a test sample including a sample materialand a wash liquid. The test sample is configured to be received withinthe sample well and the reservoir. The membrane is configured to filterthe wash liquid from the test sample during the centrifugation processsuch that the wash liquid can pass from the reservoir, through themembrane and can be captured within a collection chamber, while thesample material remains within the reservoir.

In some embodiments, a well plate includes a set of sample wells each influid communication with a reservoir from a set of reservoirs, and amembrane at least partially disposed between the set of sample wells andthe set of reservoirs. The membrane defines a set of openings that areeach in fluid communication with a different sample well from the set ofsample wells and a different reservoir from the set of reservoirs. Eachsample well from the set of sample wells and each reservoir from the setof reservoirs is configured to receive a test sample including a testmaterial and a wash liquid. The membrane is configured to filter thewash liquid from each test sample disposed within the set of samplewells and the set of reservoirs during a centrifugation process suchthat the wash liquid can pass from each reservoir from the set ofreservoirs through the membrane to be captured within a collectionchamber, while the sample material of each test sample remains withinthe respective reservoir from the set of reservoirs.

In some embodiments, a tube assembly includes a sleeve member, a toptube, a base member, and a membrane. The top tube is configured to bereceived at least partially within an interior region of the sleevemember, and the membrane is disposed between a portion of the top tubeand the base member. The top tube defines a sample well in fluidcommunication with an opening defined by the membrane and a reservoirdefined by the base member. The tube assembly is configured to be usedin a centrifugation process of a test sample, which includes a samplematerial and a wash liquid. The test sample is configured to be receivedthrough the sample well to be disposed within the reservoir. Themembrane is configured to filter the wash liquid from the samplematerial during the centrifugation process such that the wash fluid canpass up through the membrane and into a collection chamber defined bythe sleeve member, while the sample material remains within thereservoir.

Apparatus and methods are described herein for use in testing andanalysis of assays including cells, or particles similar in size tocells. In some embodiments, a device such as those described herein canbe used for assays involving cells or particles, and methods can be usedfor preparing samples by repeated washing and centrifugation, such asthose described herein. For example, a device such as those describedherein can be used with cells and/or particles that have a diameterbetween 0.5 micron and 50 microns. In one example use, any of thedevices described herein can be used to test and analyze cells and/orparticles having a diameter between 2 microns and 20 microns, which isthe size of most mammalian leukocytes.

Apparatus and methods are described herein that can be used duringcentrifugation to test sample cells and particles. Duringcentrifugation, the wash liquid used during the testing process can becaptured within a collection chamber of the apparatus or device. Thus,the need to aspirate or decant the wash liquid can be reduced oreliminated. In some embodiments, a well plate assembly can include a topplate, a bottom plate and a membrane disposed between the top plate andthe bottom plate. The well plate assembly can be used in acentrifugation process for cells and/or particles. During thecentrifugation process, while the cells are drawn to a bottom reservoirportion of the well plate assembly, the wash liquid (e.g., effluent) canrise from the reservoir, be filtered through the membrane, and directedthrough drain holes of the well plate assembly and captured in acollection chamber. In some embodiments, an absorbent member can bedisposed within the collection chamber to capture the wash liquidtherein. In some embodiments, the absorbent member can be impregnatedwith chemicals that can neutralize biohazards in the wash liquid oreffluent within the collection chamber.

As used in this specification, the singular forms “a,” “an” and “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, the term “a member” is intended to mean a singlemember or a combination of members, “a material” is intended to mean oneor more materials, or a combination thereof.

As used herein, the term “set” can refer to multiple features or asingular feature with multiple parts. For example, when referring to aset of walls, the set of walls can be considered as one wall withmultiple portions, or the set of walls can be considered as multiple,distinct walls. Thus, a monolithically constructed item can include aset of walls. Such a set of walls may include multiple portions that areeither continuous or discontinuous from each other. A set of walls canalso be fabricated from multiple items that are produced separately andare later joined together (e.g., via a weld, an adhesive, or anysuitable method).

As used herein, the term “sample” refers to a composition whose contentsand/or constituents are to be tested. A sample can be heterogeneous,containing a variety of components (e.g., different proteins) orhomogenous, containing one component. In some instances, a sample can benaturally occurring, a biological material, and/or a man-made material.Furthermore, a sample can be in a native or denatured form. In someinstances, a sample can be a single cell (or contents of a single cell)or multiple cells (or contents of multiple cells), a blood sample, atissue sample, a skin sample, a urine sample, culture media, bovineserum albumen, antibodies, cytokines, small molecule drugs, quantumdots, oligonucleotides, fluorophores, fixatives, and/or the like. Insome instances, a sample can be from a living organism, such as aeukaryote, prokaryote, mammal, human, yeast, and/or bacterium or thesample can be from a virus. In some instances, a sample can be one ormore stem cells (e.g., any cell that has the ability to divide forindefinite periods of time and to give rise to specialized cells).Suitable examples of stem cells can include but are not limited toembryonic stem cells (e.g., human embryonic stem cells (hES)), andnon-embryonic stems cells (e.g., mesenchymal, hematopoietic, inducedpluripotent stem cells (iPS cells), or adult stem cells (MSC)).

The embodiments described herein can be formed or constructed of one ormore biocompatible materials. Examples of suitable biocompatiblematerials include metals, glasses, ceramics, or polymers. Examples ofsuitable metals include pharmaceutical grade stainless steel, gold,titanium, nickel, iron, platinum, tin, chromium, copper, and/or alloysthereof. A polymer material may be biodegradable or non-biodegradable.Examples of suitable biodegradable polymers include polylactides,polyglycolides, polylactide-co-glycolides (PLGA), polyanhydrides,polyorthoesters, polyetheresters, polycaprolactones, polyesteramides,poly(butyric acid), poly(valeric acid), polyurethanes, and/or blends andcopolymers thereof. Examples of non-biodegradable polymers includenylons, polypropylene, polyesters, polycarbonates, polyethersulfone,polyacrylates, polymers of ethylene-vinyl acetates and other acylsubstituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulphonate polyolefins, polyethylene oxide, and/orblends and copolymers thereof.

FIG. 1 is a perspective view of a known well plate assembly provided forreference. FIG. 2 is a perspective view of a well plate assembly 100,according to an embodiment. FIG. 2 illustrates the well plate assembly100 with a portion removed for illustration purposes. Well plateassembly 100 (also referred to herein as “plate assembly”) can includemultiple wells 110 in which an assay can be disposed. In thisembodiment, the plate assembly 100 includes 96 wells disposed, forexample, in an 8×12 arrangement.

Well plate assembly 100 includes a top plate 120, a bottom plate 150 anda microporous membrane 140 disposed between the top plate 120 and thebottom plate 150. Top plate 120 and bottom plate 150 can be formed witha suitable material(s), such as, for example, polystyrene, nylon,polypropylene, polyethylene therethalate, polypropylene, polyethylene,polysulphone, polyethersulfone, polytetrafluoroethylene (PTFE),cellulose acetate, and/or polyvinylidene fluoride. Top plate 120 andbottom plate 150 can each be formed, for example, by an injectionmolding process, machining, 3D printing or other suitable manufacturingtechniques. In some embodiments, top plate 120 and bottom plate 150 aremonolithically formed, although it should be understood that they can beformed to include multiple components and/or multiple differentmaterials. In some embodiments, bottom plate 150 can be formed with atransparent glass material that is suitable for use with assaytechniques that require light transmission, such as, for example, withmicroscopy, while top plate 120 can be formed with a transparent plasticmaterial or, alternatively, an opaque, anti-reflective plastic material.In another example, the top plate 120 can be formed such that each well110 is formed with a highly-inert plastic material, such as, forexample, PTFE, and is molded over a support material or, alternatively,press-fit into or molded within a second plastic support material tomake top plate 120. Although this embodiment is shown as a standard 96well plate, it should be understood that the features described hereincan be scaled and applied to any other size well plate size including,for example, arrays of 4, 6, 9, 24, 384, and 1536 wells or even as asingle well tube. In some embodiments, the top plate 120 and the bottomplate 150 are separately formed by injection molding.

Membrane 140 can be formed with a substantially planar microporoussheet. In some embodiments, the membrane 140 can be formed with amaterial such that the membrane 140 is a barrier to the passage ofcells, but allows liquid to pass through with little impedance. Suchmembranes can include, but are not restricted to, hydrophilic membranesincluding those used in plasmapheresis such as polyethersulfonemembranes with a pore size between, for example, 0.2 micron and 2microns and that are considered “low-binding” in that proteins and cellsdo not readily stick to them. In some embodiments, the pore size issmaller than the diameter of the cells that are to be retained in thesample well (e.g., most leukocytes are approximately 8 microns indiameter), but large enough to allow reagents that exit the plateassembly 100 to pass through the membrane 140. For example, suchreagents can include, but are not limited to, human serum constituents,culture media, bovine serum albumen, antibodies, cytokines, smallmolecule drugs, quantum dots, oligonucleotides, fluorophores, fixatives,alcohols, and isotopes appropriate for mass cytometry chelated andattached to polymers. Additionally, the membrane 140 can be formed witha material such that the membrane 140 is chemically compatible with thereagents and wash liquids to be used in the assay process. Membrane 140may be made of a material that has low cell adhesion and otherproperties that reduce cells being bound to the membrane itself.

In some embodiments, the membrane 140 can be a hydrophilic membrane thatcan be used with aqueous wash solutions that readily wet out and reducethe effect of meniscus formation. In some embodiments, the membrane 140can be a hydrophobic membrane that can be used when it is desirable tohave low water absorption and with nonaqueous solutions. Choosing amembrane material with a high transmembrane flow rate may beadvantageous and one such membrane is a Type 6F sold by Membrana GmbH,Wuppertal, Germany, which has a transmembrane flow of greater than 90milliliters/(minute square centimeter bar). The type of membrane to usefor a particular application can also depend on the liquids to be usedin that application. For example, some assays may involve liquids thatmay interact unfavorably with the particular membrane.

In some embodiments, the membrane 140 can be formed as a porouspolypropylene membrane, which although it can be less hydrophilic canstill be used for plasmapheresis in some settings. The membrane 140 canbe formed with a variety of different materials, such as, for example,various polymers, including polypropylene, polyethylene, polysulphone,polyethersulfone, polytetrafluoroethylene, regenerated cellulose,cellulose acetate, polyvinylidene fluoride, and others.

Top plate 120 and bottom plate 150 can be coupled together byconventional methods, including those typically used to assemble filterplates. Such coupling methods can include, for example, ultrasonicwelding, insert molding, co-molding, press-fitting, thermal adhesion,and/or adhesives, such as, epoxies, urethanes and polyurethanes. Themembrane 140 can be disposed between the top plate 120 and the bottomplate 150 and be coextensive with the perimeter edges of the top plate120 and bottom plate 150, as shown, for example, in FIG. 2, or can beslightly smaller to allow the edges to be ultrasonically welded aroundthe perimeter of the top plate 120 and bottom plate 150. Alternatively,the membrane 140 may be pre-cut into an array of pieces, each largeenough for a single well. The membrane 140 can be bonded to the topplate 120, the bottom plate 150 and/or to both the top plate 120 and thebottom plate 150. In some embodiments, mechanical fasteners (not shownin FIGS. 1-14), such as, for example, screws, bolts, and/or clips can beused in addition to or alternatively to join the top plate 120 and thebottom plate 150 together. In other embodiments, the top plate 120 andthe bottom plate 150 can be formed such that they can be press-fittogether or snapped together without adhesive or fasteners.

FIG. 3 illustrates a detailed cut-away section of the top plate 120 andmembrane 140, which exposes a portion of the bottom plate 150 forillustration purposes. As shown in FIG. 3, the top plate 120 defines topopenings 130 and bottom openings 131 each in fluid communication withthe interior volume 111 of the wells 110 through which an assay can beinserted into the well 110. The top plate 120 also defines drain holes121. The membrane 140 defines openings 142 that are substantiallycentrally aligned with openings 131 of the top plate 120. The bottomplate 150 defines a reservoir 160 with a recessed portion 161 and drainholes 151. The drain holes 121 of the top plate are in fluidcommunication with drain holes 141 defined in the membrane 140, whichare each in fluid communication with one of the bottom plate drain holes151. The drain holes 121, drain holes 141 and drain holes 151 can be thesame or different diameter and can be at least partially aligned orconcentric with each other. While illustrated as generally u-shaped, thereservoir 160 and/or the recessed portion 161 can be any shape. Forexample, the reservoir 160 and/or the recessed portion 161 can beconically shaped, c-shaped, or v-shaped. In some embodiments, differentrecessed portions can have different shapes depending on, for example,the intended use of the well plate and the volume of cells to becollected, for example.

In use, in some applications where the well plate assembly 100 is to becentrifuged, a wash liquid along with cells or particles to be testedcan be disposed within an interior volume 111 (also referred to hereinas “well column”) of the wells 110. While being centrifuged, as shown byflow directional arrow F_(C) in FIG. 3, the cells are drawn to a bottomportion of the well column 111 by centripetal force, through opening 131and opening 142 and into the reservoir 160 of the bottom plate 150. Asshown by flow directional arrow F_(L) in FIG. 3, the wash liquidseparates from the cells and in an effort to meet equilibrium raises upthrough a portion of the membrane 140 to a top surface of the top plate120 between the wells 110. The wash liquid can then pass through the topplate drain hole 121, through the membrane drain hole 141, and thenthrough the bottom plate drain hole 151 and into a collection chamber(not shown) disposed, for example, below the bottom plate 150. Forexample, in some embodiments, the well plate assembly 100 can include acollection chamber or tray to collect the wash liquid that separatesfrom the cells during the centrifugation process and drops down throughthe drain holes 121, 141 and 151. The collection tray can be integratedinto a one-piece assembly by attachment to the bottom of the bottomplate 150, or can be a removable tray that can be manually emptied aftereach centrifugation step. An example of such a tray is described withrespect to well plate assemblies 300 and 400 herein (see, e.g., FIGS.17, 18 and 21).

Centripetal acceleration directly opposes the flow of liquid out of thereservoir 160 and through membrane 140, thus forcing the cells away fromthe membrane 140 and to a bottom portion of the reservoir 160. The levelof the remaining supernatant in the well column 111 can be equal to aheight of the top plate drain hole 121. The height of the entrance totop plate drain hole 121 can act as a weir, and by adjusting this heightabove the membrane 140, the amount of the remaining supernatant in thewell column 111 after centrifugation can be adjusted. The cells orparticles within the well 110 can collect or “pellet” in the reservoir160 of the bottom plate 150, and in particular, within the recessedportion 161 of the reservoir 160. The size of the pores in the membrane140 can be too small to allow the cells to pass up through the membrane140 with the wash liquid during centrifugation. The forces that bindcells and other particles irretrievably to the membrane as a consequenceof liquid flowing through the membrane must be adequately opposed by thecentripetal acceleration acting on these particles. This requiresoptimization of the device such that the well height above the membrane,reservoir geometry, membrane material, membrane pore size, andfunctional membrane surface area per well is optimized to ensure thatliquid does not flow through the membrane until sufficient centripetalacceleration is achieved and so that the rate of the flow through themembrane and other factors that would irretrievably bind particles tothe membrane are adequately opposed by centripetal acceleration.

FIG. 4 is a detailed section view of a well 110 of well plate assembly100. As shown in FIG. 4, the inner walls 112 of the wells 110 aretapered such that a diameter of the well column 111 (e.g., interiorvolume) at a base opening 131 defined by the top plate 120 is less thana diameter of the well column 111 at top opening 130. The taper of theinner walls 112 can help concentrate the cells or particles in thecentral recessed portion 161 of the reservoir 160 and or increase thefunctional surface area of membrane 140 per well. The taper can alsofacilitate the top plate 120 releasing from the mold during an injectionmolding process of forming the top plate 120.

In some embodiments, the reservoir 160 in the bottom plate 150 is alsotapered to direct the cells or particles to the center recessed portion161. The recessed portion 161 is centrally located within the lowestportion of the reservoir 160. Cells or particles can first collect inthe recessed portion 161, and depending on the quantity of cells orparticles in the sample, can also fill other areas of the reservoir 160.Such a configuration allows cells to be readily accessed (e.g., bypipetting) through the well 110, directly into the reservoir 160.

FIGS. 5-8 are further illustrations of the plate assembly 100. Theplanar perimeter portion of the top plate 120 and the bottom plate 150are not shown in FIGS. 5-8. As shown in FIG. 5, each square grouping offour wells 170 share a common top plate drain hole 121 located in thecenter of each grouping. FIG. 9 includes a perspective view of the topplate 120 and FIG. 10 is another top view of top plate 120. FIG. 11 is asection view B-B taken along line B-B in FIG. 10. FIGS. 12 and 13 are atop view and a side cross section view, respectively, of the bottomplate 150 and FIG. 14 is a top view of the membrane 140. Although plateassembly 100 includes a single membrane 140 that extends beneath allwells 110, in some embodiments, the well assembly 100 can includemultiple membranes each disposed beneath a single well 110 of the topplate 120.

FIG. 15 is an exploded view of another embodiment of a well plateassembly. A well plate assembly 200 (also referred to herein as “plateassembly”) includes a top plate 220, a microporous membrane 240, and abottom plate 250. The top plate 220, bottom plate 250, and membrane 240can be coupled together in the same or similar manner as described abovefor well plate assembly 100. Each of the top plate 120, bottom plate 150and membrane 140 can also be formed the same or similar as describedabove for well plate assembly 100.

The top plate 220 includes multiple wells 210 and defines multiple drainchannels 280 that provide a common drain to a row or group of wells 270.For example, a common drain channel 280 can be provided for a group oftwo, three, four, five, etc. wells. The supernatant that leaves thewells 210 through the membrane 240 is directed by the drain channels 280to the edge of the top plate 220 where it drops off the top plate 220and can be collected by a collection tray placed under the assembly (notshown). Drain holes as shown in assembly 100 are not required in theembodiment shown in plate assembly 200. Each of the wells 210 includeswalls 212 that define a bore with an interior volume 211. In thisembodiment, the shape of the bore is substantially rectangular. Thewells 210 also define a top opening 230 and a base opening 231 each influid communication with the interior volume 211. FIG. 16 illustrates adetailed cross section of a well 210 of well plate assembly 200. In thisembodiment, the interior walls 212 that define the interior volume 211have a constant or substantially constant shape and size (e.g., are nottapered) but could be tapered if required for ease of manufacturing. Asshown in FIGS. 15 and 10, the interior walls 212 have a cross-sectionthat is substantially rectangular.

The bottom plate 250 defines multiple reservoirs 260 disposed off-centerof the well bottom openings 230. In this embodiment, membrane 240defines multiple apertures 242 that correspond to the shape of theinterior volume 211 defined by walls 212 of top plate 220. As shown inFIG. 16, the top plate 220 defines membrane windows 221 that are eachdisposed over a portion 243 of the membrane 240 such that liquid candrain up through the membrane 240 as shown by directional arrow F_(L),through the membrane windows 221 and into drain channels 280 (shown inFIG. 15) and thereby to the outside edge of the top plate 220. Forexample, although not shown, the well plate assembly 200 can alsoinclude a collection chamber or tray to collect the wash liquid thatseparates from the cells during the centrifugation process and is passedby the drain channels 280 to the outside edge of the top plate 220. Thecollection chamber can be at least partially defined by a tray disposedbelow the bottom plate 250.

FIG. 17 shows another embodiment of a well plate assembly that issimilar in design and function to the well plate assembly 100, but withonly nine wells. A well plate assembly 300 includes a nine well array ofwells 410 and is disposed on an effluent collection tray 335. As withprevious embodiments, the well plate assembly 300 (also referred toherein as “plate assembly”) includes a top plate 320, a bottom plate 350and a membrane 340 (see, e.g., FIG. 19) disposed between the top plate320 and the bottom plate 350. The top plate 320, bottom plate 350, andmembrane 340 can be coupled together with adhesive and mechanicalfasteners 336 as shown in FIGS. 17 and 19. Each of the top plate 120,bottom plate 150 and membrane 140 can also be formed the same or similaras described above for previous embodiments.

The top plate 320 includes wells 310 that define an interior region orvolume and defines top openings 330 and bottom openings (not shown). Thetop plate 320 also includes drain holes 321. The membrane 340 includesan opening (not shown) that can be substantially aligned with the bottomopening of the top plate 320 and drain holes (not shown). The bottomplate 350 includes reservoirs (not shown) and drain holes 351, asdescribed above for previous embodiments.

As shown in FIG. 17 the well plate assembly 300 can be disposed withinan effluent collection tray (also referred to herein as “tray”) 335.FIG. 18 is a top perspective view of the effluent collection tray 335.The effluent collection tray 335 includes a containment wall 338 thatsurrounds the perimeter of the tray 335 and defines a collection chamber333 that can hold the effluent (e.g., wash liquid) that left the wellsthrough the membrane 340 during centrifugation. A central platform 339extends above a base 337 of the effluent collection tray 335 and definesan interior region 347 in which the plate assembly 300 can be disposed,as shown in FIG. 17. The platform 339 defines multiple channels 334,which allow the effluent to drain from the drain holes 321 and 351 andinto the collection chamber 333 of the tray 335.

In use, samples placed in the wells 310 can pass through bottom openings(not shown) in the top plate 320, through the apertures (not shown) ofthe membrane 340 and into the reservoirs (not shown) of the bottom plate350. During centrifugation, the cells can collect within the reservoirsof the bottom plate 350 and the effluent (e.g., wash liquid; alsoreferred to as supernatant) can rise up through the membrane 340 andthen out or down through the drain holes 321 of the top plate 320, themembrane drain holes (not shown), the bottom plate drain holes 351 andinto the collection chamber 333 of the tray 335.

FIGS. 20-22 illustrate a well plate assembly 400 that includes fourwells 410. The well plate assembly 400 (also referred to herein as“plate assembly”) includes a top plate 420, a bottom plate 450, and amembrane 440 disposed between the top plate 420 and the bottom plate450. As with the previous embodiment, the top plate 420 can be coupledto the bottom plate 450 with fasteners 490. FIG. 22 is a top view ofmembrane 440. The plate assembly 400 can be used with an effluent tray435 in a similar manner as described for plate assembly 300. Theeffluent tray 435 can include the same or similar features and functionsas described for effluent tray 335. Each well 410 of the top plate 420defines a top opening 430 and a base opening (not shown) each in fluidcommunication with an interior region 411 of the well 410. The top plate420 can also define drain holes (not shown). The base plate 450 definesfour reservoirs 460 that align with wells 410, and a drain hole 451.

The membrane 440 defines multiple well apertures 442 and multiple drainholes 441. The well apertures 442 can substantially align with the wells410. In use, samples placed in the wells 410 can pass through the bottomopening (not shown) in the top plate and through the membrane wellaperture 442 and into the reservoirs 460 of the bottom plate 450. Duringcentrifugation, the effluent can pass up through the membrane 440, andthen out through the top plate drain holes (not shown), the membranedrain hole 441, the drain holes 451 of the base plate 450 and into theeffluent tray 435.

FIGS. 23-25 illustrate another embodiment of a well plate assembly. Awell plate assembly 600 includes 96 wells 610 in a standard 8×12 arrayconfiguration. An exploded view of well plate assembly 600 is shown inFIG. 24, and FIG. 25 is an enlarged view of a portion of the well plateassembly 600 with portions removed for illustration purposes. The wellplate assembly 600 (also referred to herein as “plate assembly”)includes a top plate 620, a bottom plate 650 and a membrane 640 disposedbetween the top plate 620 and the bottom plate 650. The top plate 620includes a planar border portion or flange 622 that defines vent holes623 as shown in FIG. 25. Samples can be introduced into wells 610through a well top opening 630 defined by each of the wells 610. Thewells 610 each have interior walls 612 that define a well bore orinterior volume 611 that has a lightly-tapered top portion and aheavily-tapered bottom portion. In some embodiments, the well bore orinterior volume 611 can have a non-tapered top portion and a taperedbottom portion. The top plate 620 also defines bottom openings 631 anddrain holes 621.

The membrane 640 is a substantially planar sheet of microporoushydrophilic membrane material and defines membrane well apertures 642and drain holes 641. A vent membrane shown in 645 a and 645 b is made ofsubstantially planar sheets of a porous membrane that can filter airleaving a collection chamber (described in more detail below) whendisplaced by effluent flowing into the collection chamber. In someembodiments, the pore size of this air filtering vent membrane can be,for example, 0.2 microns. Bottom plate 650 defines drain holes 651 andwell retention reservoirs 660 with recessed portions 661, and forms anupper portion of an effluent collection chamber of a collection tray 690(described in more detail below). An optional effluent trapping membrane648 can be a substantially planar hydrophobic membrane with definedmicro-holes (not shown). An absorbent member 670 is disposed within thecollection chamber of the tray 690 and can be impregnated with chemicalsthat neutralize biohazards in the effluent within the collectionchamber.

The membrane 640 can be coupled to the top plate 620 and bottom plate650 such that a liquid-tight seal is formed between the membrane 640 andthe adjacent surfaces of the top plate 620 and the bottom plate 650.During use, cell suspensions and buffers can be loaded into the interiorvolume 611 of the wells 610 through openings 630, which define a topportion of the wells 610. In this embodiment, the wells 610 can be sizedto hold, for example, over 730 microliters of liquid. When centrifugedat a rate greater than 100 rcf, for example, the liquid flows downthrough the well openings 631, through membrane well apertures 642 andinto the well retention reservoir 660 in the bottom plate 650. Thepressure generated by the centripetal acceleration acting on the columnof liquid in the wells 610 forces the liquid up through the microporousmembrane 640. The size of the pores in the membrane 640 can be too smallto allow cells to pass through the membrane 640. Liquid that flows upthrough the membrane 640 can drain out through the top plate drain holes621, through membrane drain holes 641, through bottom plate drain holes651, through the micro-holes in effluent trapping membrane 648 (e.g.,the hydrophobic membrane), and lastly into the absorbent material member670 within the collection tray 690 where any biohazardous agents (suchas, for example, viral or bacterial pathogens, etc.) in the effluent canbe neutralized by chemicals in the absorbent material member 670 (e.g.,formaldehyde, sodium hypochlorite, etc.).

After the height of the liquid in the wells 610 is no higher than thelevel of the top plate drain holes 621, the pressure generated bycentripetal acceleration acting on the liquid in the wells 610 is nolonger sufficient for liquid to pass through membrane 640. In thisembodiment, the volume of liquid that remains in the well 610 (e.g., theresidual volume) is slightly greater than the volume of the wellretention reservoir 660, which can be, for example, 90 microliters.During centrifugation, the centripetal acceleration of well plateassembly 600 can pull the cells away from the membrane 640 and oppositethe flow of the liquid through the membrane 640, and the cells cancollect in the recessed portions 661 of reservoirs 660. This canminimize cells sticking to the membrane 640 and thereby minimizes cellloss, as well as membrane clogging.

The effluent trapping membrane 648 (e.g., hydrophobic membrane withmicro-holes) is an optional component of the well plate assembly 600.The effluent trapping membrane 648, if employed, can function as aone-way valve such that effluent enters the collection chamber (definedby the bottom plate 650 and the tray 690) from the drain holes 651 inthe bottom plate 650 driven by the centripetal acceleration generatedduring centrifugation, but cannot pass back through the drain holes 651to the top plate 620 during normal handling, even if inverted, as theforce of gravity alone is insufficient to make polar liquids passthrough small diameter micro-holes in a hydrophobic membrane. Duringnormal handling, effluent membrane 648 can help prevent liquids in theabsorbent material member 670 from coming to the surface of the topplate 620 and possibly contaminating adjacent wells 610 by travelingback through membrane 640 and into the well collection reservoirs 660.The hydrophobic membrane has micro-holes aligned with the drain holes inthe top plate. The sizes of the micro-holes are sufficient to allowefficient transfer of effluent into the collection chamber duringcentrifugation including any macromolecules in the effluent, but theirsmall diameter coupled with the hydrophobic nature of the membraneprohibits polar liquids from passing back through during normalhandling. The size of these micro-holes can be determined by the desiredfunction and can in some embodiments be, for example, between 0.2microns and 1000 microns.

The vent membrane 645 a and 645 b filters air displaced from thecollection chamber to reduce or limit biohazardous elements from exitingthe collection chamber as aerosol or as airborne particles.

FIGS. 26-28 illustrate a tube assembly 715, according to an embodiment.Similar to well plate assembly 600 described above, a tube assembly canbe employed for certain types of assays in which it is desirable tocollect and neutralize potentially hazardous effluent. As shown, forexample, in FIG. 26, the tube assembly 715 includes a top tube 720 thatdefines a top opening 730. The top tube 720 includes a flange 724 thatrests on a sleeve flange 726 and allows for limited insertion of toptube 720 within a sleeve member 725. A microporous membrane 740 isdisposed between top tube 720 and a base 750. Liquid can pass up throughmembrane 740, through a notch 721 in the top tube 720, through a notch741 in the membrane 740, then through a notch 751 in the base 750, andinto a bottom portion of sleeve member 725. An optional absorbentmaterial member 770 can rests within the bottom portion of sleeve member725 and can absorb any effluent.

As shown, for example, in FIG. 26, in this embodiment, top tube 720 oftube assembly 720 includes a tapered inner bore 712 in fluidcommunication with top opening 730 and a base opening 731. Membrane 740has a circular tube aperture 742 centrally disposed and aligned with thetube base opening 731. Samples can be placed into tube 720 and can flowinto a reservoir 760 of the base 750, and cells or particles can collectin recessed portions 761 of the reservoir 760. During centrifugation,liquid flows through membrane 740 and eventually into the bottom portionof sleeve 725 where it can be absorbed by absorbent material member 770.

FIGS. 29-33 illustrate data generated during, for example, use of thefour-well plate assembly 400 described herein. Specifically, a prototypeof well plate assembly 400 was used to process human blood samples andgenerate the data shown in FIGS. 29-33. After washing, the samples wererun in BD TruCount™ tubes to obtain the most accurate cell number countspossible. FIG. 29 includes three bivariate dot plots summarizing thedata from flow cytometric analysis of a Propidium Iodide (PI) washoutexperiment demonstrating the efficacy of the prototype washing cellsfollowing staining with PI. PI is a fluorescent dye that binds to DNAand efficiently stains permeabilized cells making these cells fluorescewith well-known excitation and emission frequencies. Use of PI foranalytical purposes requires that cells remain in a solution of PIbecause washing PI stained cells has been shown to reduce the amount ofPI bound to the cells (PI washout). In FIG. 29 methanol permeabilizedperipheral blood mononuclear cells (PBMC) from a human whole bloodsample were analyzed by a flow cytometer prior to staining with PI (PlotA), after staining with PI (Plot B), and after washing the stained cellswith the prototype (Plot C). Individual cells are represented asindividual dots on the plots where their position in two dimensionalspace is plotted by their intrinsic light scattering property on thex-axis (linear scale) and the amount of PI fluorescence is plotted onthe y-axis (logarithmic scale, base 10). Where multiple cells overlap inthe plot a color scale from blue through green to red allows thevisualization of the density of cells in that region of the plot. Plot Ashows the methanol permeabilized peripheral blood mononuclear cells(PBMC) from the blood sample prior to staining with PI; note the dots(cells) are plotted relatively low on the y-axis because there is nosignal from PI and the detector is registering only background noise inthe PI detection channel (PerCP-Cy55 channel). Plot B shows thepermeabilized PBMC after staining with PI, but before washing with theprototype; note the cells are plotted relatively high on the y-axisbecause there is so much PI bound to the cells that it is near themaximum value that can be registered on the detection channel. Plot Cshows the permeabilized and PI stained PBMC after washing with theprototype; note the cells are plotted much lower on the y-axis than inPlot B corresponding to a significant reduction in the amount of PIbound to the cells. The y-axis in these plots is on a logarithmic base10 scale so washing in the prototype results in a greater than 50 foldreduction in PI fluorescence.

The results of a surface gating of whole blood phospho-flow experimentwashed in the prototype of well plate assembly 400 is shown in FIG. 30and the intracellular staining from that experiment is shown in FIG. 31.This experiment demonstrates the utility of this well plate assemblydesign for antibody staining and washing samples for complex flowcytometry experiments. A human whole blood sample was split into twoaliquots, one incubated for 15 minutes with cytokine interferon alpha(IFNa) at a final concentration of 100 nanograms per milliliter whilethe other aliquot was incubated with a similar volume of phosphatebuffered saline as a control. At the end of the 15 minute incubationboth aliquots were fixed, erythrocytes lysed, and antibody staining fordetection of surface epitopes was followed by methanol permeabilizationand subsequent antibody staining for detection of intracellular epitopesas known in the art (see, e.g., U.S. Patent App. Publication No.2009/0155838, the disclosure of which is incorporated herein byreference in its entirety). Plot D shows a population of cells highlypositive for cell surface marker CD66 which corresponds to granulocytesand much lower levels of this marker on other cells types, as expected.It also shows a clearly CD33 positive population that is low for CD66expression which corresponds to Monocytes. The population that is lowfor both CD66 and CD33 is analyzed in Plot E for expression of CD3 andCD4; two populations are found to be positive for CD3 corresponding toCD4+ and CD4− T cells, as expected. Taking all of the CD3 positive cellsand analyzing them in the bivariate Plot F it is clear that both theCD4+ and CD4− populations of T cells have their own subsets of CD45RA+and CD45RA− T cells corresponding to naïve and memory/effector T cells,respectively, as known in the art. Plots on FIG. 31 show theintracellular phospho-specific antibody staining results from thisexperiment for three different cell types defined by the expression ofsurface epitopes listed at the top of each plot (gating based on theseepitopes as shown in FIG. 30); Plot G shows the phospho-specificantibody staining for Naïve CD4+ T cells, Plot H shows thephospho-specific antibody staining for Memory/Effector T cells, and PlotI shows the phospho-specific antibody staining for Monocytes. Incubatinghuman PBMC with IFNa is known in the art to induce phosphorylation ofspecific amino acid residues (specific phospho-epitopes) on thesignaling proteins STAT1 and STAT3. In this experiment, twofluorescently labeled antibodies that bind with high specificity tothese specific phospho-epitopes were used to stain the cells. Cells fromthe aliquot incubated with IFNa are plotted as red dots (labeled R inFIG. 31), cells from the aliquot incubated with the saline control areplotted as black dots (labeled B in FIG. 31). As expected, in each PlotG, H, and I the red dots are to the right and above the black dotscorresponding to significant detection of IFNa induced phosphorylationof STAT1 and STAT3. An unwashed fraction was diluted four times prior tobeing run, which helps reduce background noise.

FIG. 32 shows the effectiveness of the well plate assembly 400 reducingbackground staining of surface antibodies when the well plate assemblywas used to wash stained cells. In an ideal scenario, where thebackground binding of the antibody was zero, the intensity of theantibody's staining on cells lacking the target surface protein would bezero. In actual use, antibodies do not bind only to their target butalso other elements of the cell, referred to as non-specific staining,and as a result the background staining of every antibody is distinctlynon-zero, especially when cells are fixed and permeabilized. Backgroundstaining is one of the primary challenges of flow cytometry, especiallyintracellular flow cytometry, because it limits signal-to-noise andthereby makes it difficult to detect low-abundance analytes in cells.Washing cells has been shown to effectively reduce the backgroundbinding of all antibodies by washing away a high fraction of antibodiessticking non-specifically to the cell while not dislodging an antibodymolecule if bound to its proper target due to the high-affinity of thisinteraction. In each Plot J, K, and L a histogram shows the amount ofbackground staining found on each cell type (each cell type defined by acombination of surface markers listed at the top of the plot) where thex-axis shows the level of staining with fluorescently labeled antibodythat has high specificity for a surface marker not found on the celltype in that plot. The y-axis scale is from zero to one hundred percentand quantifies the fraction of the sample at each value on the x-axis.The x-axis value of the peak of each histogram is approximately themedian value of background staining (x-axis value) for the cell type.The red histogram (labeled R in FIG. 32) in each plot shows thebackground staining of the cells prior to being washed in the prototypeand the blue histogram (labeled B in FIG. 32) shows the backgroundstaining of the cells after being washed in the prototype. As can beseen in each Plot J, K, and L, the peak of the blue histogram isdistinctly to the left of the peak of the red histogram indicating thatwashing in the prototype significantly reduced background staining. Notethat the x-axis is a logarithmic scale, base 10, so the difference inthe peaks corresponds to approximately a 5 fold difference in backgroundstaining. Washing was effective even when large protein fluorophoressuch as phycoerythrin (PE) were conjugated to the antibodies. Though notshown here, washing was effective even when Quantum Dots were conjugatedto the antibodies demonstrating that the membrane of the well plateassembly allowed Quantum Dot conjugates to pass through (e.g., the poresize was large enough).

FIG. 33 shows the effectiveness of the washing process on backgroundstaining with phospho-specific antibodies. All plots are of an aliquotof human blood that was incubated with saline as a control so the cellshave low levels of the phospho-epitopes targeted by the phospho-specificantibodies used here. The cell surface markers that identify the celltype are listed at the top of each column of plots. The histograms inthe top row of plots quantify the staining of the cells with an antibodythat binds to a specific tyrosine residue of the signaling protein STAT3only when it is phosphorylated (tyrosine 705). The histograms in thebottom row of plots quantify the staining of the cells with an antibodythat binds to a specific tyrosine residue of the signaling protein STAT1only when it is phosphorylated (tyrosine 701). As in FIG. 32, the redhistogram (labeled R in FIG. 33) in each plot shows the backgroundstaining of the cells prior to being washed in the prototype and theblue histogram (labeled B in FIG. 33) shows the background staining ofthe cells after being washed in the prototype. Staining permeabilizedcells with these antibodies results in some non-specific backgroundbinding to the cells such that histograms of the cells prior to beingwashed by conventional methods is higher than after being washed byconventional methods. As can be seen in the plots of FIG. 33, the bluehistograms are to the right of the red histograms in each plotdemonstrating that the prototype effectively washes cells stained withphospho-specific antibodies and thereby reduces background staining withthese antibodies.

In another example, a well plate assembly as described herein can bedesigned to optimize cell counting by flow cytometry (CD4 T cell countsfor HIV patients as well as other applications). In this example, eachwell of the well plate assembly has within it a known number of beadswhose light scattering and fluorescence make them easy to discriminatefrom leukocytes (similar to the approach used by Becton Dickinson'sTruCount tubes). Within the well are also the desired fluorescentlylabeled antibodies required to stain the blood samples such that cellsof interest are properly characterized by expression of cell surfacemarkers that are specifically bound by the antibodies (i.e., CD3, CD4,CD19, CD14, CD66, etc.). For maximal antibody stability, the antibodiesmay be in a solution containing agents that improve their stability suchas bovine serum albumin or the antibodies may be lyophilized orotherwise dried down (the beads can also be dried down). An appropriatevolume of the whole human blood to be analyzed is added to the wells ofthe well plate assembly and incubated at 4 degrees Celsius for 20minutes until antibody staining is complete. An appropriate volume wouldbe, for example, 100 microliters, but much smaller and much largervolumes are also anticipated including, but not limited to volumesbetween 5 microliters up to 1 milliliter. If desired, erythrocyte lysiscan be carried out after this 20 minute antibody staining reaction byhypotonic shock through the addition of distilled water or ammoniumchloride erythrocyte lysis buffer, or other techniques known in thefield for erythrocyte lysis. A wash buffer composed of phosphatebuffered saline (with or without the addition of bovine serum albuminand or other reagents) is added in a volume sufficient to stop the lysisreaction.

The well plate assembly is then centrifuged to generate a centripetalacceleration that permits proper operation (e.g., 100 rcf to 200 rcf maybe sufficient, but higher or lower accelerations may be desired forproper function) such that most of the liquid fraction of the sample(the effluent) flows through membrane 1 (e.g., membrane 648 in wellplate assembly 600—the hydrophilic membrane), then drains through thedrain holes, through the micro holes in membrane 2 (e.g., membrane 648in well plate assembly 600—the hydrophobic membrane), which are alignedwith the drain holes, and into the absorbent material in the collectiontray where any biohazardous agents in the effluent are neutralized bychemicals in the absorbent material (such as, for example, concentratedsodium hypochlorite). The stained, lysed, and washed samples can then beanalyzed by flow cytometry or by other cell based analysis methods. Anapparatus as described herein can be configured such that it can be useddirectly on flow cytometers or other analysis instruments capable ofdrawing samples directly from 96 well plates. Although some of theseinstruments are not currently configured to draw samples from such adeep well plate, optional accessories for such devices can allow the useof deep welled plates.

Referring now to FIGS. 34-41, a well plate assembly 800 (also referredto herein as “plate assembly”) and a tray assembly 895 are illustratedaccording to an embodiment. The tray assembly 895 can be any suitableshape, size, or configuration. For example, as shown in FIGS. 34-37, thetray assembly 895 can include a coupling tray 835, a collection tray855, and a drain tray 885 arranged in a substantially stackedconfiguration. The coupling tray 835 can be configured to couple thewell plate assembly 800 to the tray assembly 895. More specifically, thecoupling tray 835 includes containment walls 838 that define an innervolume 829 that can receive a portion of the well plate assembly 800.Furthermore, opposite sides of, for example, two of the containmentwalls 838 can include and/or define a coupling portion 832 that canselectively engage a portion of the well plate assembly 800 such thatthe well plate assembly 800 traverses the inner volume 829. In thismanner, a portion of the well plate assembly 800 can be disposed in theinner volume 829 (e.g., the portion of the well plate assembly 800 issubstantially circumscribed by the containment walls 838). Thearrangement of the coupling tray 835 can be such that the couplingportion 832 can engage a coupling portion of any number of well plateassemblies 800 having any suitable configuration. For example, as shownin FIGS. 34-37, the coupling portion 832 can be configured to matinglyengage coupling portions or flanges 822 of the well plate assembly 800.Although a single well plate assembly 800 is shown, more than one wellplate assembly 800 can be coupled to coupling tray 835. For example, asshown in FIG. 34, in this embodiment, the well plate assembly 800includes 16 wells, and six well plate assemblies 800 can be coupled tothe coupling tray 835. In other embodiments, the coupling portion 832can be configured to engage a well plate assembly including, forexample, 96 wells or more. The tray assembly 895 can also include and/orcan be coupled to a lid 814 that is configured to substantially coverand/or enclose the inner volume 829 of the coupling tray 835 and/or thewell plate assembly 800 coupled thereto.

As shown in FIG. 35, the coupling tray 835 includes a base surface 828that defines a drain portion 827. The base surface 828 can be anysuitable configuration. For example, in some embodiments, the basesurface 828 can be separated into, for example, distinct portions thatcan have a slope between the containment walls 838 and the drain portion827. Said another way, in some embodiments, the base surface 828 can beseparated into sections that each form an obtuse angle (e.g., greaterthan 90°) with a corresponding inner surface of the containment walls838. Said yet another way, the base surface 828 can be divided intoportions that are each angled away from the containment walls 838towards the drain portion 827 such that the drain portion 827 isdisposed, for example, below a bottom surface of the containment walls838. In this manner, a fluid can flow within the inner volume 829 andinto contact with a portion of the base surface 828, and with the basesurface 828 being divided into substantially sloped sections, the fluidcan flow along the slope of the portion of the base surface 828 towardsthe drain portion 827. As shown in FIGS. 35 and 36, the drain portion827 can be substantially open (e.g., the drain portion 827 can includeand/or can define one or more openings). Thus, a fluid can flow along aportion of the base surface 828 towards the drain portion 827, and canflow substantially out of the inner volume 829 of the coupling tray 835via the drain portion 827.

As shown in FIGS. 35 and 36, the coupling tray 835 of the tray assembly895 can be coupled to and/or otherwise disposed adjacent to thecollection tray 855. The collection tray 855 can be any suitableconfiguration. For example, as shown in FIG. 36, the collection tray 855can define an inner volume 833 (e.g., a collection chamber). As such,when the collection tray 855 is coupled to the coupling tray 835 (asshown in FIG. 35), a fluid that flows through, for example, the drainportion 827 of the coupling tray 835 can be collected, stored, retained,and/or the like in the inner volume 833 of the collection tray 855. Thecollection tray 855 can include a valve ball 856, and a bias member 857.Furthermore, as shown, the collection tray 855 can include a valve seat858 and define an opening 859. As such, when the collection tray 855 iscoupled to and/or otherwise disposed adjacent to the coupling tray 835,a first end portion of the bias member 857 can be placed in contact witha surface of the coupling tray 835 and a second end portion (e.g., anopposite end portion) can be in contact with the ball valve 856. Theball valve 856, in turn, can be in contact with the valve seat 858included in the collection tray 855. Thus, the bias member 857 (e.g., aspring or the like) can be configured to exert a biasing force thatsubstantially maintains the ball valve 856 in contact with the valveseat 858. In some instances, this arrangement can fluidically isolatethe opening 859 from the inner volume 833 (e.g., the collection chamber)in the absence of an externally applied force (e.g., a force exerted tomove the ball valve 856 relative to the valve seat 858, as described infurther detail herein.

As shown in FIGS. 34, 35, and 37, the collection tray 855 of the trayassembly 895 can be coupled to and/or otherwise disposed adjacent to thedrain tray 885. Said another way, the tray assembly 895 can be arrangedsuch that the collection tray 855 is disposed between the coupling tray835 and the drain tray 885. More specifically, as shown in FIGS. 34 and37, the drain tray 885 can include and/or can be coupled to a retainingclip 884. The retaining clip 884 can be any suitable configuration. Forexample, as shown in FIG. 37, the retaining clip 884 can be asubstantially planar plate from which a set of clip arms 883 can extend.The clip arms 883 can be any suitable configuration. As such, when theretaining clip 884 is coupled to the drain tray 885 and the drain tray885 is placed adjacent to the collection tray 855, the clip arms 883 canengage a portion or surface of the coupling tray 835 to couple thecollection tray 855 and the drain tray 885 thereto (e.g., the clip arms883 can include a protrusion, tab, latch, etc. that can engage, forexample, a recess, tab, notch, etc. of the coupling tray 835).

The drain tray 885 can be any suitable configuration. For example, asshown in FIG. 37, the drain tray 885 can include a drain protrusion 887,a set of walls 888, and a recessed surface 886. The recessed surface 886defines a channel 880 that is in fluid communication with an opening 889defined by the walls 888, as described in further detail herein. In someembodiments, the channel 880 can include, for example, a flexible hoseor the like. As such, the flexible hose can be connected to a laboratoryvacuum trap or similar device for removing the liquid that collects inthe recessed surface 886 in a manner that is considered safe andconvenient in the laboratory environment. For example, in someinstances, a vacuum trap and/or the like can be used to aspiratesupernatant from the recessed surface 886, filter plates, and/or similardevices. Moreover, the recessed surface 886 can be substantiallynon-planar. For example, in a similar manner as described above with thecoupling tray 835, the arrangement of the drain tray 885 can be suchthat the recessed surface 886 forms a slope away from the walls 888towards the drain protrusion 887. Thus, a fluid can flow along a portionof the recessed surface 886 towards the drain protrusion 887. The drainprotrusion 887 can extend from a central portion of the recessed surface886 to selectively engage a portion of the collection tray 855. Morespecifically, the arrangement of the drain tray 885 and the collectiontray 855 can be such that, when disposed adjacent to one another, thedrain protrusion 887 extends through the opening 859 of the collectiontray 855 and into contact with the ball valve 856. As such, when thecollection tray 855 is disposed between the coupling tray 835 and thedrain tray 885 and the drain tray 885 is coupled o the coupling tray835, the drain protrusion 887 can exert an external force on the ballvalve 856 that is sufficient to overcome a force exerted by the biasmember 857 and thus, the ball valve 856 can be moved away from the valveseat 858. In this manner, the opening 859 can place the inner volume 833(e.g., the collection chamber) in fluid communication with the draintray 885. Furthermore, the arrangement of the channel 880 can be suchthat at portion of the channel substantially circumscribes an area ofthe drain protrusion 887. Therefore, a fluid can flow from, for example,the well plate assembly 800, through the drain portion 827 of thecoupling tray 835, through the opening defined by the collection tray855, into, for example, the flexible tubing guided by the channel 880and through the opening 889 to be collected in a laboratory vacuum trapand/or the like, as described in further detail herein.

As shown in FIGS. 38-41, the plate assembly 800 includes a top plate 820(also referred to herein as “top portion”), a membrane 840, and a bottomplate 850 (also referred to herein as “bottom portion”). The top plate820 can be at least temporarily coupled to the bottom plate 850 suchthat the membrane 840 is disposed therebetween. The top plate 820includes coupling portions or flanges 822 disposed on opposite sides ofthe plate assembly 800 that can be operable in at least temporarilycoupling the plate assembly 800 to the coupling portion 832 of thecoupling tray 835, as described above. For example, the couplingportions 822 can define openings 844 that can receive therethrough tabs846 of the coupling portions 832. As shown in FIGS. 34 and 35, the plateassembly 800 can be coupled to a first section of the coupling portion832 (e.g., not across the entire coupling portion 832) and extendacross, for example a width of the coupling tray 835. As such, anysuitable number of plate assemblies 800 can be coupled to the couplingportion 832 such as, for example, one plate assembly, two plateassemblies, three plate assemblies, four plate assemblies, five plateassemblies, six plate assemblies, or more. As shown in FIG. 34, sixplate assemblies 800 are coupled to the tray assembly 895.

The top plate 820 can include and/or can define a set of wells 810 thatcan receive therein a sample (e.g., a biological sample) to be tested,for example, using a centrifugal testing system or the like. Morespecifically, the top plate 820 includes a set of inner walls 812 thatcan form at least a portion of a boundary defining the wells 810. Theinner walls 812 can have any suitable arrangement. That is to say, thetop plate 820 can define any suitable number of wells 810 in anysuitable arrangement. For example, as shown in FIG. 38, the arrangementof the inner walls 812 can be such that the top portion 820 defines aset of 16 wells 810 in a 2×8 well arrangement. More particularly, thearrangement of the inner walls 812 can be such that the set of wells 812are defined in 2×8 grid arrangement, with each well 810 defining and/orencompassing a volume of substantially equal size and/or shape. Thearrangement of the inner walls 812 can also be such that the top plate820 defines a set of four drain columns 816 (e.g., collection columns orthe like), with each drain column 816 being in selective fluidcommunication with a subset of four wells 810, as described in furtherdetail herein.

Although, the arrangement of the plate assembly 800 is specificallydescribed as including the 16 wells 810 in a 2×8 arrangement, in otherembodiments, the plate assembly 800 can include and/or can define anynumber of wells 810 in any suitable arrangement. For example, in someembodiments, a plate assembly can include and/or can define 96 wells inan 8×12 arrangement. In still other embodiments, a plate assembly caninclude and/or can define any suitable number of wells in any suitablearrangement (e.g., one well, two wells, four wells, six wells, ninewells, 24 wells, 48 wells, 128 wells, 144 wells, 384 wells, 768 wells,1536 wells, and/or any number of wells therebetween. Similarly, althoughthe plate assembly 800 is specifically described as including four draincolumns 816, in other embodiments, a plate assembly can include anynumber of drain columns that can be configured to be in selective fluidcommunication with any number of wells such that each well included inthe plate assembly is in fluid communication with at least one draincolumn.

Expanding further, as shown in FIGS. 38 and 41, the arrangement of theinner walls 812 of the top plate 820 is such that the wells 810 aregrouped, subdivided, and/or otherwise collected into subsets disposedaround or about one of the drain columns 816. For example, the set ofwells 810 can be grouped into subsets of four wells, with each well in aset being in fluid communication with one drain column 816 associatedwith that set of four wells. More specifically, the arrangement of thetop portion 820 can be such that a portion of the inner walls 812defining each well 810 included in a subset (e.g., a grouping of fourwells) also forms and/or defines the drain column 816 and vice versa. Inother words, the arrangement of the top plate 820 can be such that aportion of the inner walls 812 that defines a drain column 816 alsodefines a portion of each of the four wells 810 in the subset disposedabout the drain column 816. For example, as shown in FIGS. 38 and 41, insome embodiments, a portion of the inner walls 812 can form and/or canbe arranged as a substantially cylindrical annulus to define the drainvolumes 816 therebetween (i.e., internal of the substantiallycylindrical annulus), and a portion of the wells 810 thereabout (i.e.,external of the substantially cylindrical annulus). Furthermore, aportion the inner walls 812 that forms the substantially cylindricalannulus can define an opening, port, channel, and/or the like that canselectively place the wells 810 included in the subset of wells 810disposed about that drain column 816 in fluid communication with atleast one drain column 816, as described in further detail herein.Although the inner walls 812 are specifically described above asdefining the wells 810 and the drain columns 816, the arrangement of thetop plate 820 can also be considered as including any number of wells810 and any number of drain columns 816, each of which has an outerstructure or the like that can form, for example, a portion of the innerwalls 812 of the top plate 820.

The wells 810 and/or the inner walls 812 defining the wells 810 caninclude and/or can define a top opening 830 and a bottom opening 831, asshown in FIG. 41. As such, a sample can be delivered to the well 810 viathe top opening 830 and the well 810 can be configured such that thesample can pass therethrough via the bottom opening 831. Moreover, theinner walls 812 include a tapered portion 813 such that a size (e.g.,diameter) of the bottom opening 831 is smaller than a size of the topopening 830. Expanding further, the arrangement of the wells 810 and/orthe inner walls 812 can be such that a channel 819 is defined between anouter surface of the tapered portion 813 and, for example, an innersurface of a portion of the inner walls 812 defining that well 810. Thechannel 819 can be configured to substantially circumscribe the taperedportion 813 of the inner walls 812 (and thus, a portion of thecorresponding well 810). Moreover, as shown in FIG. 41, the inner walls812 can define an opening 818 that can place the channel in fluidcommunication with the drain column 816. In addition, the plate assembly800 can include a divider 817 or the like that is disposed within thedrain column 816. The divider 817 can, for example, divide and/orseparate the drain column 816 into substantially fluidically isolatedportions. As such, the inner walls 812 and/or the drain column 816 candefine a set of the openings 818 with each opening being associated witha divided segment or volume of the drain column 816. Thus, a flow of afluid, liquid, and/or the like can flow through the channel 819associated with, for example, a first well 810 and into a first opening818 to be disposed in a first portion of the drain column 816, and aflow of a fluid, liquid, and/or the like can flow through a channel 819associated with, for example, a second well 810 and into a secondopening 818 to be disposed in a second portion of the drain column 816.

Although not shown in FIGS. 38-41, in some embodiments, a bottom surfaceand/or portion of the top plate 820 can be arranged such that thechannel 819 extends therethrough (e.g., the bottom surface of the topplate 820 defines an opening corresponding to the channel 819 associatedwith each well 810). As described in further detail herein, thearrangement of the plate assembly 800 can be such that when the membrane840 is disposed adjacent to and in alignment with the top plate 820, themembrane 840 substantially covers the channel 819 (e.g., the channel 819is physically separated from a volume on an opposite side of themembrane 840).

The membrane 840 of the plate assembly 800 is configured to be disposedbetween the top plate 820 and the bottom plate 850 of the plate assembly800 (see e.g., FIG. 41) when the top plate 820 and the bottom plate 850are coupled together. The membrane 840 can be formed from and/or can bearranged as a substantially planar microporous sheet. In someembodiments, the membrane 840 can be formed with a material such thatthe membrane 840 is a barrier to the passage of cells, but allows liquidto pass through with little impedance. Such membranes can include, butare not restricted to, hydrophilic membranes including those used inplasmapheresis such as polyethersulfone membranes with a pore sizebetween, for example, 0.2 micron and 2 microns and that are considered“low-binding” in that proteins and cells do not readily stick to them.In some embodiments, the pore size is smaller than the diameter of thecells that are to be retained in the sample well (e.g., most leukocytesare approximately 8 microns in diameter), but large enough to allowreagents to pass through the membrane 840. For example, such reagentscan include, but are not limited to, human serum constituents, culturemedia, bovine serum albumen, antibodies, cytokines, small moleculedrugs, quantum dots, oligonucleotides, fluorophores, fixatives,alcohols, and isotopes appropriate for mass cytometry chelated andattached to polymers. Additionally, the membrane 840 can be formed fromand/or can include a material that is chemically compatible with thereagents and wash liquids to be used in the assay process. As such, themembrane 840 can be formed from any suitable material and/or can haveany suitable arrangement such as those described above with reference tothe membrane 140 in FIGS. 1-14.

As shown in FIG. 39, the membrane 840 defines a set of drain openings841 and a set of well openings 842. The arrangement of the plateassembly 800 can be such that when the membrane 840 is disposed betweenthe top plate 820 and the bottom plate 850 (e.g., when the top plate 820is coupled to the bottom plate 850), the drain openings 841 aresubstantially aligned with and/or are coaxial with a corresponding draincolumn 816 and the well openings 842 are substantially aligned withand/or are coaxial with a corresponding well 810. Thus, the arrangementof the drain openings 841 and the well openings 842 can allow a sampleand/or a portion thereof to pass through the membrane 840, as describedin further detail herein. Although shown in FIG. 39 as being asubstantially continuous membrane 840 having a size and/or shape that isassociated with an overall size and/or shape of the top portion 820, inother embodiments, the membrane 840 can include any number ofindependent portions with each portion being, for example, associatedwith and/or substantially aligned with a different well 810, asdescribed above with reference to the membrane 140.

The bottom plate 850 of the plate assembly 800 includes an inner surfacethat defines and/or forms multiple reservoirs 860 each having a recessedportion 861, as shown in FIG. 40. In addition, the bottom plate 850defines a set of drain openings 851. As described above, the bottomplate 850 is configured to be coupled to the top plate 820. In someembodiments, the bottom plate 850 can be removably coupled to the topplate 820 (e.g., via a friction fit, a press fit, a snap fit, amechanical fastener, a latch, a threaded coupling, and/or the like orcombination thereof). In other embodiment, the bottom plate 850 can befixedly coupled to the top plate 820 (e.g., via an adhesive such as, anepoxy, a urethane, a polyurethane, etc.; via a mechanical coupling suchas, a friction fit, a press fit, ultrasonic welding, friction welding;via any other suitable manufacturing process such as, insert molding,over-molding, co-molding, thermal adhesion, etc.; and/or the like or acombination thereof).

As shown in FIG. 41, the bottom plate 850 can be coupled to the topplate 820 such that the fluid reservoirs 860 are substantially alignedwith and/or coaxial with a corresponding well 810 of the top plate 820(i.e., each fluid reservoir 860 is aligned with a different well 810),while the drain openings 851 are substantially aligned with and/orcoaxial with a corresponding drain column 816 (i.e., each drain opening851 is aligned with a different drain column 816). With the membrane 840disposed between the top plate 820 and the bottom plate 850 and with themembrane 840 substantially aligned with the top plate 820 (as describedabove), the drain openings 851 of the bottom plate 850 can also bealigned with and/or coaxial with the drain openings 841 defined by themembrane 840 and the reservoirs 860 of the bottom plate 850 can also besubstantially aligned with and/or coaxial with the well openings 842defined by the membrane 840. Said another way, the arrangement of theplate assembly 800 (e.g., when the membrane 840 is disposed between thetop plate 820 and the bottom plate 850 and the top plate 820 and thebottom plate 850 are coupled together, as shown in FIG. 41) can be suchthat each well 810 (and more specifically, the bottom opening 831 ofeach well 810) shares an axial centerline A_(W) with a correspondingwell opening 842 of the membrane 840 and a corresponding reservoir 860of the bottom portion 850. Similarly, each drain column 816 (and moreparticularly, a drain opening 821 associated with each drain column 816)shares an axial centerline A_(d) with a corresponding drain opening 841of the membrane 840 and a corresponding drain opening 851 of the bottomplate 850. In addition, the plate assembly 800 can be arranged such thatthe membrane 840 physically separates the reservoir 860 from the channel819 circumscribing the tapered portion 813 of the inner walls 812defining the corresponding or aligned well 810. Thus, with the membrane840 being selectively permeable, the membrane 840 can allow, forexample, a fluid disposed in the reservoir 860 to pass through themembrane 840 to be disposed in the channel 819 and in turn, with thechannel 819 in fluid communication with the drain column 816 (via theopening 818 described above), the fluid can pass through the channel 819to be disposed in the drain column 816.

In use, a sample can be delivered through the top opening 830 of anysuitable number of wells 810 defined by the plate assembly 800. Underthe force of gravity, at least a portion of the sample, can flow throughthe well 810 and can exit the wells 810 via the bottom openings 831.With the membrane 840 and the bottom plate 850 aligned with the topplate 820, as described above, a portion of the sample can similarlyflow through the corresponding well openings 842 of the membrane 842 tobe disposed in the corresponding reservoirs 860. With the samplesdisposed in the plate assembly 800, the plate assembly 800 can becoupled to the tray assembly 895 (e.g., via the coupling portion 832 ofthe coupling tray 835 and the flange 822 of the top portion 820 of theplate assembly 800) and the lid 814 can be disposed about the couplingtray 835. The tray assembly 895 can then be disposed in, for example, acentrifuge device. Although the sample is described above as beingtransferred to the wells 810 prior to the plate assembly being coupledto the tray assembly 895, in other instances, the plate assembly 800 canbe coupled to the tray assembly 895 and subsequently, the sample can betransferred to the wells 810. In such instances, the sample can betransferred to the wells 810 prior to the tray assembly 895 beingdisposed in the centrifuge device or after being disposed in thecentrifuge device.

While being centrifuged, as shown by the flow directional arrow F_(C) inFIG. 41, the remaining portion of the sample in well 810 is drawn to abottom portion of the well 810 and through the openings 831 and 842defined by the top plate 820 and the membrane 840, respectively, andinto the reservoir 860 of the bottom plate 850. As shown by flowdirectional arrow F_(L) in FIG. 41, the wash liquid separates from thecells and is urged to flow through a portion of the membrane 840 andinto the channel 819 defined by the inner walls 812. More specifically,as the tray assembly 895 is rotated, the sample disposed in thereservoir 860 is exposed to centripetal force and/or acceleration aswell as centrifugal force and/or acceleration. As such, the trayassembly 895 can be rotated with sufficient rotational velocity toseparate the sample, for example, by density. In this manner, thecentrifugal force and/or acceleration exerted on portions of the samplehaving a lower density (e.g., less dense portions such as, for example,liquid portions including wash liquids, fluids, or the like) can besufficient to overcome, for example, the centripetal forces similarlyexerted on the portions of the sample, as well as, for example, afriction force between the portions of the sample and the inner surfaceof the bottom plate 850 defining the reservoir 860. Thus, thecentrifugal force exerted on the portions of the sample can urge suchportions (e.g., the portions having a lower density) to flow along thesurface of the reservoir 860 (radially and/or in an expanding spiralaway from, for example, the axial centerline A_(W)) towards the membrane840. Moreover, the centrifugal force exerted on, for example, suchfluids (e.g., the less dense portions of the sample) can be sufficientto urge the fluids to flow through the membrane 840 and into the channel819. The portion of the sample (e.g., the wash liquid, effluent, etc.)can then pass through the opening 818 and into the drain column 816. Thefluids can then flow through the drain hole 821 defined by the top plate820, through the drain hole 841 defined by the membrane 840, and throughthe drain hole 851 of the bottom plate 850, thereby flowing out of theplate assembly 800. Although not shown in FIGS. 34-41, in someembodiments, the inner volume 833 of the collection tray 855 and/or anyother suitable portion of the tray assembly 895 can include an absorbentand/or trapping material that is configured to absorb and/or neutralize,for example, a biohazardous fluid or the like as described above forprevious embodiments.

With the plate assembly 800 coupled to the tray assembly 895, the fluidscan flow along the base surface 828 of the coupling tray 835, throughthe drain portion 827, and into the inner volume 833 (e.g., thecollection chamber) defined by the collection tray 855. Furthermore,with the drain protrusion 887 engaging the ball valve 856, the fluidscan flow through the opening 859 and into the channel 880 defined by thedrain tray 885. The arrangement of the drain tray 885 is such that thefluids can then flow within the channel 880 and through the opening tobe discarded in any suitable biologically safe manner.

As described above, centripetal force and/or acceleration directlyopposes the flow of liquid out of the reservoir 860 and through membrane840, thus forcing the cells away from the membrane 840 and into therecessed portion 861 of the reservoir 860. The level of the remainingsupernatant in the well 810 can be equal to a height of the drain hole821 of the top plate 820. The height of the entrance to drain hole 821can act as a weir, and by adjusting this height above the membrane 840,the amount of the remaining supernatant in the well 810 aftercentrifugation can be adjusted. The cells or particles within the well810 and/or the reservoir can collect or “pellet” in, for example, therecessed portion 861 of the reservoir 860. Moreover, as described above,the size of the pores in the membrane 840 can be sufficiently small asto not allow the cells to pass up through the membrane 840 with the washliquid during centrifugation. In this manner, centrifugal processes canbe serially performed on the sample with substantially less sample lossthan would otherwise be possible.

Referring now to FIGS. 42-51, a well plate assembly 900 (also referredto herein as “plate assembly”) and a tray assembly 995 are illustratedaccording to another embodiment. The tray assembly 995 can be anysuitable shape, size, or configuration. As shown in FIGS. 42 and 43, thetray assembly 995 can include a coupling tray 935, a collection tray955, and a drain tray (not shown) arranged in a similar manner asdescribed above for tray assembly 895. As shown, for example, in FIG.45, the tray assembly 995 can include and/or can be coupled to a lid 914or the like that can substantially cover, for example, the plateassembly 900 when the plate assembly 900 is coupled to the tray assembly995. The tray assembly 995 can be substantially similar to the trayassembly 895 described above with reference to FIGS. 34-37. Thus, thediscussion of the tray assembly 895 can apply to the tray assembly 995unless explicitly expressed otherwise and hence, the tray assembly 995is not described in further detail herein.

As shown in FIGS. 42-51, the plate assembly 900 includes a top plate 920(also referred to herein as “top portion”), a membrane 940, and a bottomplate 950 (also referred to herein as “bottom portion”). The top plate920 can be at least temporarily coupled to the bottom plate 950 suchthat the membrane 940 is disposed therebetween. In some embodiments,portions of the plate assembly 900 can be substantially similar to orthe same as corresponding portions of the plate assembly 800 describedabove with reference to FIGS. 34-41. Thus, some aspects of the plateassembly 900 are not described in further detail herein and should beconsidered the same as the corresponding aspects of the plate assembly800 unless explicitly expressed otherwise.

The top plate 920 can include and/or can define multiple wells 910 thatcan receive therein a sample (e.g., a biological sample) to be tested,for example, using a centrifugal testing system or the like. Morespecifically, the top plate 920 includes a set of inner walls 912 thatcan form at least a portion of a boundary defining the wells 910. Theinner walls 912 can have any suitable arrangement. That is to say, thetop plate 920 can define any suitable number of wells 910 in anysuitable arrangement. For example, as shown in FIGS. 42 and 46-49, thearrangement of the inner walls 912 can be such that the top portion 920defines a set of 96 wells 910 in a 12×8 well arrangement. Thearrangement of the inner walls 912 can also be such that the top plate920 defines a set of 24 drain columns 916 (e.g., collection columns),with each drain column 916 being in fluid communication (i.e., via themembrane 940) with a subset of wells 910 (e.g., four wells), asdescribed above with reference to the plate assembly 800. In otherwords, liquid can pass through the membrane 940 and into the draincolumn 916. As shown, for example, in FIGS. 42 and 43, the top plate 920includes a set of flanges 922 disposed on opposite sides of the plateassembly 900 that can be operable in at least temporarily coupling theplate assembly 900 to a coupling portion 932 of the coupling tray 935.Expanding further, although the plate assembly 800 included a singleflange 822 on opposite sides of the top plate 820 that engaged a portionof the coupling portion 832, the plate assembly 900 can include, forexample, six flanges 922 disposed on opposite sides of the top plate 920that can engage substantially the entire extent of the coupling portion932 of the coupling tray 935 to couple the plate assembly 900 to thetray assembly 995.

As shown in FIGS. 42-43, 48, and 49, the arrangement of the inner walls912 of the top plate 920 is such that the wells 910 are grouped,subdivided, and/or otherwise collected into subsets disposed around orabout one of the drain columns 916. For example, the set of wells 910can be grouped into subsets of four wells, with each well in the subsetbeing in fluid communication (i.e., via the membrane 940) with one draincolumn 916 associated with that set of four wells, as described indetail above with reference to the top plate 820 in FIGS. 38 and 41. Thewells 910 and/or the inner walls 912 defining the wells 910 can includeand/or can define a top opening 930 and a bottom opening 931, as shownin FIG. 44. As such, a sample can be delivered to the well 910 via thetop opening 930 and the well 910 can be configured such that the samplecan pass therethrough via the bottom opening 931. Moreover, the innerwalls 912 include a tapered portion 913 such that a size (e.g.,diameter) of the bottom opening 931 is smaller than a size of the topopening 930. Expanding further, the arrangement of the wells 910 and/orthe inner walls 912 can be such that a channel 919 is defined between anouter surface of the tapered portion 913 and, for example, an innersurface of a portion of the inner walls 912 defining that well 910. Thechannel 919 can substantially circumscribe the tapered portion 913 ofthe inner walls 912 (and thus, a portion of the corresponding well 910).Moreover, as shown in FIG. 41, the inner walls 912 can define an opening918 that can place the channel 919 in fluid communication with the draincolumn 916, as described in detail above with reference to the top plate820 in FIGS. 38 and 41.

The membrane 940 of the plate assembly 900 is configured to be disposedbetween the top plate 920 and the bottom plate 950 of the plate assembly900 (see e.g., FIGS. 46 and 47) when the top plate 920 and the bottomplate 950 are coupled together. The membrane 940 can be formed fromand/or can be arranged as a substantially planar microporous sheet. Insome embodiments, the membrane 940 can be formed with a material suchthat the membrane 940 is a barrier to the passage of cells, but allowsliquid to pass through with little impedance, such as those describedabove with reference to the membrane 840 in FIG. 39. As shown in FIGS.46 and 47, the membrane 940 defines a set of drain openings 941 and aset of well openings 942. The arrangement of the plate assembly 900 canbe such that when the membrane 940 is disposed between the top plate 920and the bottom plate 950 (e.g., when the top plate 920 is coupled to thebottom plate 950), the drain openings 941 are substantially aligned withand/or are coaxial with a corresponding drain column 916 and the wellopenings 942 are substantially aligned with and/or are coaxial with acorresponding well 910. Thus, the arrangement of the drain openings 941and the well openings 942 can allow a sample and/or a portion thereof topass through the membrane 940, as described in further detail herein.Moreover, the membrane 940 can have a size and shape that is associatedwith, for example, the 96 well configuration of the top plate 920.

The bottom plate 950 of the plate assembly 900 includes an inner surfacethat defines and/or forms a reservoir 960 having a recessed portion 961,as shown in FIGS. 46, 47, 50, and 51. In addition, the bottom plate 950defines a set of drain openings 951. As described above, the bottomplate 950 is configured to be coupled to the top plate 920 via anysuitable coupling method. As shown in FIGS. 44, 46, and 51, the bottomplate 950 can be coupled to the top plate 920 such that the fluidreservoirs 960 are substantially aligned with and/or coaxial with acorresponding well 910 of the top plate 920 (i.e., each fluid reservoir960 is aligned with a different well 910), while the drain openings 951are substantially aligned with and/or coaxial with a corresponding draincolumn 916 (i.e., each drain opening 951 is aligned with a differentdrain column 916). With the membrane 940 disposed between the top plate920 and the bottom plate 950 and with the membrane 940 substantiallyaligned with the top plate 920 (as described above), the drain openings951 of the bottom plate 950 can also be aligned with and/or coaxial withthe drain openings 941 defined by the membrane 940 and the reservoirs960 of the bottom plate 950 can also be substantially aligned withand/or coaxial with the well openings 942 defined by the membrane 940,as described above with reference to the plate assembly 800 in FIGS.34-41. In addition, the plate assembly 900 can be arranged such that themembrane 940 physically separates the reservoir 960 from the channel 919circumscribing the tapered portion 913 of the inner walls 912 definingthe corresponding or aligned well 910. Thus, with the membrane 940 beingpermeable, the membrane 940 can allow, for example, a fluid disposed inthe reservoir 960 to pass through the membrane 940 to be disposed in thechannel 919 and in turn, with the channel 919 in fluid communicationwith the drain column 916 (via the opening 918 described above), thefluid can pass through the channel 919 to be disposed in the draincolumn 916.

In use, a sample can be delivered through the top opening 930 of anysuitable number of wells 910 defined by the plate assembly 900. Underthe force of gravity, at least a portion of the sample can flow throughthe well 910 to be disposed in the corresponding reservoirs 960, asdescribed in detail above with reference to the plate assembly 800. Withthe samples disposed in the plate assembly 900, the plate assembly 900can be coupled to the tray assembly 995 (e.g., via the coupling portion932 of the coupling tray 935 and the flange 922 of the top portion 920of the plate assembly 900) and the lid 914 can be disposed about thecoupling tray 935 (see e.g., FIG. 45). The tray assembly 995 can then bedisposed in, for example, a centrifuge device. Although the sample isdescribed above as being transferred to the wells 910 prior to the plateassembly being coupled to the tray assembly 995, in other instances, theplate assembly 900 can be coupled to the tray assembly 995 andsubsequently, the sample can be transferred to the wells 910. In suchinstances, the sample can be transferred to the wells 910 prior to thetray assembly 995 being disposed in the centrifuge device or after beingdisposed in the centrifuge device.

While being centrifuged, the remaining portion of the sample in well 910is drawn to a bottom portion of the well 910 and into the reservoir 960of the bottom plate 950. As described above, the centrifugation of thesample, can be such that the wash liquid separates from the cells byvirtue of the greater density of the cells, and as such, the pressuregenerated by centripetal acceleration acting on the column of liquid inwell 910 is sufficient for the liquid to be propelled through a portionof the membrane 940 and into the channel 919 defined by the inner walls912. The wash liquid can then pass through the opening 918 (FIGS. 47 and51) and into the drain column 916. As shown, for example, in FIGS. 43and 44, the plate assembly 900 can include a set of caps 913 or the likethat can each be disposed in a drain column 916 that can, for example,prevent the wash liquid from passing over the divider 917 and therebygenerating the risk of contaminating the channel 919 of adjacent wells.Contaminating liquid from adjacent wells 910 entering the channel 919could result in the contamination of the sample in the reservoir 960 ofthat well 910 in the event that contaminating liquid flows in thereverse direction through the membrane 940 when the liquid levels in thewell 910 and in the channel 919 have reached near equilibrium. Althoughthe caps 913 are shown as being separate components, in otherembodiments, the caps 913 can be formed, for example, as part of the topplate 920. For example, the caps 913 can be formed integral with thedividers 917 and/or otherwise formed by the walls 912 of the top plate920.

As such, the wash liquid can then flow through the drain hole 921defined by the top plate 920, through the drain hole 941 defined by themembrane 940, and through the drain hole 951 of the bottom plate 950,thereby flowing out of the plate assembly 900. With the plate assembly900 coupled to the tray assembly 995, the wash liquid and/or effluentcan flow through a drain portion 927 of the coupling tray 935 and intoan inner volume 933 (e.g., the collection chamber) defined by thecollection tray 955. While the drain tray is not shown in FIGS. 42-51,in some instances, a drain protrusion of the drain tray can engage aball valve 956 (see e.g., FIG. 44), and the wash liquid can flow throughan opening, which is otherwise obstructed by the ball valve 956, andremoved by, for example, a flexible hose disposed in and/or defining achannel of the drain tray. The arrangement of the drain tray (not shown)is such that the wash liquid can then flow within the channel andthrough an opening to be discarded in any suitable biologically safemanner.

As described above, centripetal force and/or acceleration directlyopposes the flow of liquid out of the reservoir 960 and through membrane940, thus forcing the cells away from the membrane 940 and into therecessed portion 961 of the reservoir 960. Thus, the cells or particleswithin the well 910 and/or the reservoir 960 can collect or “pellet” in,for example, the recessed portion 961 of the reservoir 960 (see e.g.,FIG. 51). Moreover, as described above, the size of the pores in themembrane 940 can be sufficiently small as to not allow the cells to passup through the membrane 940 with the wash liquid during centrifugation.In this manner, centrifugal processes can be serially performed on thesample with substantially less sample loss than would otherwise bepossible. Although the method of using the plate assembly 900 and thetray assembly 995 in a centrifugal process for testing has been brieflydescribed, it should be understood that, while, the plate assembly 900includes and/or defines more wells 910 than the plate assembly 800, thecentrifuge process described above with reference to the plate assembly800 can apply to a similar centrifuge process of the plate assembly 900.Thus, the centrifuge process was not described in detail above.

Although the plate assembly 900 has been particularly shown anddescribed above, in other embodiments, a plate assembly can beconfigured to include and/or define a set of wells, openings,reservoirs, channels, walls, etc. in any suitable arrangement. Forexample, while the plate assembly 900 is shown in FIGS. 48-51 asincluding the top plate 920 in which the wells 910 are substantiallysquare and the drain column 916 is substantially round, in otherembodiments, a plate assembly can include a top plate with any suitableconfiguration. Similarly, while the plate assembly 900 is shown in FIGS.48-51 as including the bottom plate 920 having, for example,substantially V-shaped reservoirs 960, in other embodiments, a plateassembly can include a bottom plate with any suitable configuration. Forexample, FIGS. 52-54 illustrate a portion of a well plate assembly 1000according to another embodiment. The portion of the well plate assembly1000 (also referred to herein as “plate assembly”) can be substantiallysimilar in form and function as the plate assembly 900 described abovewith reference to FIGS. 42-51. Thus, similar portions of the plateassembly 1000 and/or its use are not described in further detail herein.The plate assembly 1000 can differ from the plate assembly 900, however,in the arrangement of, for example, a top plate 1020 and a bottom plate1050. For example, as shown in FIG. 54, while the bottom plate 950 wasshown and described as defining reservoirs 960 with a substantiallyV-shaped cross-sectional area, the bottom plate 1050 defines multiplereservoirs 1060 with a substantially U-shaped cross-sectional area.Accordingly, as shown in FIGS. 52 and 53, the top plate 1020 of the wellplate assembly 1000 can have a set of wells 1010 that define a bottomopening 1031 that is substantially larger than the bottom opening 931defined by the top plate 920. Moreover, the bottom opening 1031 can havea size and/or diameter such that the bottom opening 1031 issubstantially unobstructed by a drain column 1016.

FIGS. 55-57 illustrate a portion of a well plate assembly 1100 accordingto another embodiment. The portion of the well plate assembly 1100 (alsoreferred to herein as “plate assembly”) can be substantially similar inform and function as the plate assembly 900 described above withreference to FIGS. 42-51. Thus, similar portions of the plate assembly1100 and/or its use are not described in further detail herein. Theplate assembly can differ from the plate assembly 900, however, in thearrangement of, for example, a top plate 1120 and/or a bottom plate1150. For example, as shown in FIGS. 55 and 56, while the top plate 920is shown and described above as including a set of wells 910, each ofwhich were in fluid communication with a drain column 916, the top plate1120 defines a set of wells 1110 that are each in fluid communicationwith two distinct drain columns 1116 and 1116′. Thus, in a similarmanner as described above, each well 1110 of the well plate assembly1100 can receive a sample and can undergo a centrifugal process suchthat a wash liquid or the like of the sample passes through a membrane(not shown) and into the drain column 1116 or the drain column 1116′. Asshown in FIG. 57, in some embodiments, the bottom plate 1150 defines aset of reservoirs 1160. In some embodiments, the bottom plate 1150 canbe substantially similar to or the same as the bottom plate 950described above with reference to, for example, FIG. 50.

FIGS. 58-60 illustrate a portion of a well plate assembly 1200 accordingto another embodiment. The portion of the well plate assembly 1200 (alsoreferred to herein as “plate assembly”) can be substantially similar inform and function as the plate assembly 900 described above withreference to FIGS. 42-51. Thus, similar portions of the plate assembly1200 and/or its use are not described in further detail herein. Theplate assembly 1200 can differ from the plate assembly 900, however, inthe arrangement of, for example, a top plate 1220 and/or a bottom plate1250. For example, as shown in FIGS. 58 and 59, while the top plate 920is shown and described above as including a set of wells 910 that areeach substantially square, the top plate 1220 defines a set of wells1210 that are each substantially cylindrical. Thus, in a similar manneras described above, each well 1210 of the well plate assembly 1200 canreceive a sample and can undergo a centrifugal process such that a washliquid or the like of the sample passes through a membrane (not shown)and into a drain column 1216. As shown in FIG. 60, the bottom plate 1250defines a set of reservoirs 1260 that can have a substantially broadU-shaped cross-sectional area that can, for example, correspond to thecylindrical wells 910.

Although the top plates 920, 1020, 1120, and 1220 are shown anddescribed above as including a set of drain columns 916, 1016, 1116, and1216, respectively, that have a substantially cylindrical shape (i.e.,circular cross-sectional shape when viewing top-down), in otherembodiments, a top plate can define a set of drain columns having anysuitable top-down cross-sectional shape. For example, FIG. 61illustrates a top plate 1320 of a well plate assembly according toanother embodiment. The top plate 1320 can be substantially similar inform and function as the top plate 920 described above with reference toFIGS. 42-51. Thus, similar portions of the plate assembly 1320 and/orits use are not described in further detail herein. The plate assembly1320 can differ from the plate assembly 920, however, in the arrangementof, for example, a set of wells 1310 and/or a set of drain columns 1316.For example, as shown in FIG. 61, while the top plate 920 is shown anddescribed above as including a set of drain columns 916 that aresubstantially cylindrical and/or annular, the top plate 1320 defines aset of drain columns 1316 that have a hexagonal cross-sectional shape,which in turn, can modify at least one surface defining each well 1310.In this manner, the top plate 1320 can function in a substantiallysimilar manner as described above with reference to the top plate 920.

By way of another example, FIG. 62 illustrates a top plate 1420 of awell plate assembly according to another embodiment. The top plate 1420can be substantially similar in form and function as the top plate 920described above with reference to FIGS. 42-51. Thus, similar portions ofthe plate assembly 1420 and/or its use are not described in furtherdetail herein. The plate assembly 1420 can differ from the plateassembly 920, however, in the arrangement of, for example, a set ofwells 1410 and/or a set of drain columns 1416. For example, as shown inFIG. 62, while the top plate 920 is shown and described above asincluding a set of drain columns 1416 that are substantially cylindricaland/or annular, the top plate 1420 defines a set of drain columns 1416that have a substantially diamond-shaped cross-sectional shape, which inturn, can modify at least one surface defining each well 1410. In thismanner, the top plate 1420 can function in a substantially similarmanner as described above with reference to the top plate 920.

By way of another example, FIGS. 63-65 illustrate a well plate assembly1500 according to another embodiment. The well plate assembly 1500includes a top plate 1520, a bottom plate 1550, and a membrane 1540. Incontrast to top plate 920 described above, the top plate 1520 defines aset of wells 1510, yet does not include a set of drain columns (e.g.,such as the drain columns 916). For example, as shown in FIGS. 63 and65, the top plate 1520 defines a set of side openings 1518 defined by,for example, a side of the top plate 1520 that can allow a fluid (e.g.,a wash liquid) to drain from the well plate assembly 1520. For example,wash liquid that has passed through the membrane 1540 can drain out theside openings 518 and drain downward to be collected.

The top plate 1520 and bottom plate 1550 can be coupled together suchthat the membrane 1540 is disposed therebetween, as described in detailabove with reference to the well plate assembly 800 of FIGS. 34-41. Eachwell 1510 of the top plate 1520 can define a bottom opening 1531, witheach bottom opening 1531 being substantially aligned with and/or coaxialwith a corresponding opening 1542 defined by the membrane 1540 (seee.g., FIGS. 63 and 65). As such, each well 1510 can be in fluidcommunication with a reservoir 1560 defined by the base plate 1550, in asimilar manner as described in detail above with reference to, forexample, the well plate assembly 800. Moreover, the arrangement of thetop plate 1520 can be such that the top plate 1520 defines channels 1519with each channel 1519 substantially circumscribing a portion of acorresponding well 1510. As shown in FIG. 65, each channel 1519 is influid communication with a side opening 1518 of the top plate 1520.Thus, as described in detail above with reference to other embodiments,the well plate assembly 1500 can be used in a centrifugal process suchthat cells of a sample remain disposed, for example, in a recessedportion of the reservoirs 1560 (e.g., due to centripetal acceleration)and a fluid (e.g., a wash liquid) separates from the cells (e.g., thesample) and is urged to pass up through the membrane 1540 (e.g., due tocentrifugal acceleration). The fluid can then be urged to flow throughthe corresponding channels 1519 and the corresponding side openings 1518to exit the well plate assembly 1500. In some embodiments, the fluid canexit the well plate assembly 1500 and pass through a tray assembly suchas the tray assembly 895 described above with reference to FIGS. 34-41.

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, and notlimitation. Where schematics and/or embodiments described above indicatecertain components arranged in certain orientations or positions, thearrangement of components may be modified. While the embodiments havebeen particularly shown and described, it will be understood thatvarious changes in form and details may be made. Although variousembodiments have been described as having particular features and/orcombinations of components, other embodiments are possible having acombination of any features and/or components from any of embodiments asdiscussed above. Furthermore, any portion of the apparatus and/ormethods described herein may be combined in any combination, exceptmutually exclusive combinations. While certain embodiments have beendescribed in detail above, it should be understood that variousembodiments can share common features and such description appliesequally to such features between embodiments. The embodiments describedherein can include various combinations and/or sub-combinations of thefunctions, components, and/or features of the different embodimentsdescribed.

Where methods and/or events described above indicate certain eventsand/or procedures occurring in certain order, the ordering of certainevents and/or procedures may be modified. Additionally, certain eventsand/or procedures may be performed concurrently in a parallel processwhen possible, as well as performed sequentially as described above.

What is claimed is:
 1. A method, comprising: disposing a test samplewithin a sample well of a well plate, the well plate including a topplate, a bottom plate defining a reservoir, and a membrane disposedbetween the top plate and the bottom plate, the test sample including awash liquid and a sample material; centrifuging the test sample suchthat a centripetal acceleration associated with the centrifuging drawsthe sample material away from the membrane and opposes a binding of thesample material to the membrane, the membrane configured to filter thewash liquid from the test sample during the centrifuging such that thewash liquid can pass from the reservoir defined by the bottom plate inthrough a first portion of the membrane in a direction that directlyopposes the centripetal acceleration during centrifuging, then in asecond direction perpendicular to the first direction, then in a thirddirection opposite the first direction through a second portion of themembrane different than the first portion and is captured within acollection chamber in fluid communication with the reservoir via themembrane, such that wash liquid passes from the reservoir and throughthe membrane, while the sample material remains within the reservoir. 2.The method of claim 1, wherein the wash liquid moves in the seconddirection laterally in a plane of the membrane.
 3. The method of claim1, wherein the wash liquid filtered through the membrane leaves thereservoir during the centrifuging without requiring subsequentaspiration of excess wash liquid from the sample well of the sampleplate.
 4. The method of claim 1, wherein the wash liquid filteredthrough the membrane leaves the reservoir during centrifuging withoutrequiring removal of a supernatant trapping element from the sample wellof the well plate.
 5. The method of claim 1, wherein the collectionchamber is disposed substantially beneath the well plate and coupled tothe bottom plate of the well assembly.
 6. The method of claim 1, whereinthe collection chamber is disposed substantially beneath the well plateand removably coupled to the bottom plate of the well assembly.
 7. Themethod of claim 1, further comprising: absorbing at least a portion ofthe filtered wash liquid collected within the collection chamber with atrapping membrane disposed within the collection chamber.
 8. The methodof claim 1, further comprising: absorbing at least a portion of thefiltered wash liquid collected within the collection chamber with atrapping membrane disposed within the collection chamber; andneutralizing biohazards within the filtered wash liquid captured withinthe collection chamber.
 9. The method of claim 1, wherein the samplematerial remaining within the reservoir forms a pellet.
 10. A method,comprising: disposing each of a plurality of test samples within adifferent sample well from a plurality of sample wells of a well plate,the well plate including a top plate, a bottom plate and a membranedisposed between the top plate and the bottom plate, the bottom platedefining a plurality of reservoirs, each sample well from the pluralityof sample wells in fluid communication with a reservoir from theplurality of reservoirs, each test sample from the plurality of testsamples including a wash liquid and a sample material; centrifuging theplurality of test samples such that a centripetal accelerationassociated with the centrifuging draws the sample material within eachsample well away from the membrane and opposes a binding of each samplematerial to the membrane, the membrane configured to filter the washliquid from the test sample in each sample well from the plurality ofsample wells during the centrifuging such that the wash liquid can passfrom a respective reservoir from the plurality of reservoirs through afirst portion of the membrane in a first direction that directly opposesthe centripetal acceleration during centrifuging, then in a seconddirection perpendicular to the first direction, then in a thirddirection opposite the first direction through a second portion of themembrane different than the first portion and is captured within acollection chamber in fluid communication with each reservoir from theplurality of reservoirs via the membrane, such that wash liquid passesfrom each respective reservoir and through the membrane, while eachsample material remains within the respective reservoir from theplurality of reservoirs.
 11. The method of claim 10, wherein the washliquid moves in the second direction laterally in a plane of themembrane.
 12. The method of claim 10, wherein the wash liquid from eachsample well from the plurality of sample wells that is filtered throughthe membrane leaves the respective reservoir from the plurality ofreservoirs during the centrifuging without requiring subsequentaspiration of excess wash liquid from the respective sample well fromthe plurality of sample wells of the sample plate.
 13. The method ofclaim 10, wherein the wash liquid from each sample well from theplurality of sample wells that is filtered through the membrane leavesthe respective reservoir from the plurality of reservoirs duringcentrifuging without requiring removal of a supernatant trapping elementfrom the respective sample well from the plurality of sample wells ofthe well plate.
 14. The method of claim 10, wherein the collectionchamber is disposed substantially beneath the well plate and coupled tothe bottom plate of the well assembly.
 15. The method of claim 10,wherein the collection chamber is disposed substantially beneath thewell plate and removably coupled to the bottom plate of the wellassembly.
 16. The method of claim 10, further comprising: absorbing atleast a portion of the filtered wash liquid collected within thecollection chamber with a trapping membrane disposed within thecollection chamber.
 17. The method of claim 10, further comprising:absorbing at least a portion of the filtered wash liquid collectedwithin the collection chamber with a trapping membrane disposed withinthe collection chamber; and neutralizing biohazards within the filteredwash liquid captured within the collection chamber.
 18. The method ofclaim 10, wherein the sample material remaining within each reservoirfrom the plurality of reservoirs forms a pellet.